Once genetically conditioned in the research labs of Point Prometheus and Fontaine Futuristics, a Big Daddy's main purpose is to wander Rapture, searching for a Little Sister to protect, while she is gathering ADAM. Once paired with a Little Sister, the Big Daddy follows her like a faithful dog and fiercely fights off anyone who approaches or attacks her. A standard Big Daddy has limited mental abilities and no free will. They exist as docile "sleepwalkers" who will fight to the death if their Little Sister is threatened or if they themselves are attacked. Some Big Daddies can be seen doing repairs to the city. Both the Rosie and Bouncer Big Daddy types from the first game return, and three new ones are introduced: the Rumbler, the Alpha Series, and the Lancer (exclusive to Minerva's Den).
The Big Sisters are Little Sisters who have passed through puberty. Overexposure to ADAM throughout their lives has had major effects on their mental stability, and caused them to be extremely violent. The ADAM also grants them immense strength, agility and the use of powerful Plasmids, making them formidable enemies. They have the appearance of thin, feminine versions of Big Daddies, possessing similar looking diving suits and equipment. They also have cages on their backs which can be used to carry Little Sisters. When the player incurs a Big Sister's wrath, they will have a limited amount of time to prepare before she attacks. Each Big Sister has a large ADAM needle on her left arm which is used for precision melee attacks and to drain ADAM from live victims, allowing her to heal. Big Sisters are also able to jump across an entire room.
Little Sisters continue to wander the halls of Rapture, extracting blood from corpses, and processing it through their bodies to recover the ADAM. Many of the Little Sisters in the city were taken from the surface, as revealed through the course of There's Something in the Sea. Little Sisters are protected by Big Daddies, and Subject Delta has the ability to "Adopt" these Little Sisters to collect ADAM from corpses. Delta can also 'Harvest' or 'Rescue' them. After Subject Delta deals with a certain number of Little Sisters, a Big Sister will appear to attack him for his interference.
'Splicer' is a term for the genetically altered citizens of Rapture, who became aggressive lunatics after their minds and bodies were distorted by unchecked genetic manipulation through the use of ADAM. Once addicted to ADAM, a Splicer's primary concern is procuring more. They see anyone unfamiliar as a potential target, and will attack for a chance to gain a little ADAM, including Little Sisters protected by Big Daddies. As with the first game, five types of Splicers are encountered: Thuggish, Leadhead, Spider, and Houdini, with a new type, the Brute, appearing instead of the Nitro.
Hostile Security Bots, Security Cameras, and Turrets will target the player on sight. In BioShock 2 there is a new type of Turret. It is a smaller, portable kind that can be carried around (see Mini-Turret). These are used as weapons by the Rumbler Big Daddy. They are also useable by the player. Many Vending Machines are security protected and can call Security Bots when a hacking attempt on them fails.