Bacteria growing in the oral cavity are an important effective factor in causing caries. Molars are the most commonly affected by bacterial decay. Bacterial species and their counts in caries of the second mandibular molar compared with those in caries-free teeth were investigated. A total of 100 patients with caries in the second mandibular molar and 95 caries-free individuals participated in a case–control study. A swab from the molar zone was cultured to identify the bacterial species. Colony counts and the frequency with which isolated bacteria occur were also determined. The isolated bacteria comprised 34 species ranging between 22 Gram-positive and 12 Gram-negative bacteria from caries molar and 14 species from the control group were diagnosed. Staphylococcus aureus and Kocuria rosea were revealed to have a more significant frequency in caries patients, whereas Kocuria kristinae was isolated with a high frequency from caries-free individuals. The majority of isolates had a large number of colonies, mostly Gram-positive bacteria. Some species have been found in one group of participants but not in another. Six bacterial species were only isolated from control group, whereas the patient group had 16 species that were not found in the control group. In conclusion, Gram-positive bacteria are most frequently associated with caries on the second mandibular molar. Some species of Gram-positive bacteria such as S. aureus and K. rosea and Gram-negative bacteria such as Pseudomonas fluorescens and Escherichia coli occur with great diversity and quantity with caries of the second mandibular molar compared with that of healthy individuals. Meanwhile, some species may disappear or be limited to the decayed area of the second mandibular molar.