Dental caries is the localized destruction of dental tissues by bacterial activity; the carious lesion is the result of demineralization of enamel-and later of dentine by acids produced by plaque microorganisms as they metabolize dietary carbohydrates. This study was conducted in two schools in Khartoum state, aimed to isolate and identify bacterial and fungal pathogens from dental caries infected school children. A total of 50 male students were enrolled in this study. The age ranging from 6 to 14 years, with mean of 10.9 + and 2.5 SD. (62%) of participants said they suffer from tooth pain and 19 (38%) of them visited the dentist before. According to eating habits majority of the students 48 (96°) drink milk, 47 (94%) eat sweets/candy, 38 (76%) eat crisps and 35 (70%) take soft drink. Samples were cultured and isolated bacterial and fungal pathogen were identified microscopically and by biochemical tests. Culture results were as follows: 41(82%) of samples showed bacterial and fungal growth, of those positive culture 34 (68%) showed bacterial growth, 1 (2%) fungal growth and 6 (12%) mixed growth (both bacterial and fungal). Streptococcus mutans 27(54%) was the predominant bacteria followed by Enterococcus faecalis 11(22%), Lactobacillus. spp 1(2%), and staphylococcus aureus 1(2%). Seven of the specimens (14%) showed growth of candida albicans. There was no statistically significant association between age/consumption of sweet food and microbial isolation. Significant association was detected between microbial growth and brush change (P. value = 0.041) but not with frequency of teeth brushing. In conclusion, Streptococcus mutans was the predominant bacterium isolated from caries lesions, followed by Enterococcus faecalis. The disease was mostly caused by bacteria, with only one type of fungus, Candida albicans, being isolated. No association was found between the microbial cultures and age, sugar intake, or oral hygiene. The frequency of brush change has significant association with microbial isolation.