Dental caries is one of the most prevalent and costly diseases that are a challenge to health care providers. There is limited data on the bacterial profile and risks associated with dental caries in Nigeria. The study aimed to isolate and identify bacteria and risk factors associated with dental caries in Enugu. It was a case-controlled study. A total of 336 samples were randomly collected comprising of 125 healthy subjects and 211 patients with dental caries attending various hospitals and dental clinics in Enugu, Nigeria. Samples were subjected to standard microbiological and biochemical techniques. Standardized questionnaires were used to record demographic variables and risk factors. A total of 635 and 254 microorganisms were isolated from carious lesions and healthy subjects respectively. Of these 635 isolates, 218 (34.4%) Gram-positive bacilli, 210 (33.0%) Gram-positive cocci, 46 (7.2%) Gram-negative cocci, and 43 (6.8%) yeasts were recovered. Two hundred and seven samples were (98.1%) polymicrobial while 4 samples (1.9%) were monomicrobial. Among the organisms recovered, anaerobic Lactobacillus spp (141, 66.8%) was the most prevalent followed by Streptococcus mutans (104, 49.2%), and the least were Actinomyces israelii and Capnocytophaga spp (1, 0.05%) respectively. Of the 254 isolates from apparently healthy subjects, Streptococcus sanguis ranked highest and the least was Porphyromonas gingivalis. The factors that were found to be significantly associated with dental caries were level of education P=0.005, feeding habit P= 0.001, Favourite snack P=0.004, sweet consumption P< 0.0001, type of gum P < 0.0001, history of toothache P < 0.0001, visit dentist P< 0.0001, and the bleeding gum P < 0.0001. This research highlighted the polymicrobial nature of carious lesions and associated predictors of dental caries. Hence public enlightenment and the implementation of oral health education in schools are crucial.