In view of the morbidity potential of oral complications in patients with leukemia, this study evaluated the clinical and microbiological alterations that occur in the oral mucosa of children with acute lymphoblastic leukemia (ALL) undergoing antineoplastic chemotherapy and prophylactic administration of 0.12% chlorhexidine gluconate. The sample consisted of 17 children aged 2 to 12 years that underwent clinical examination of the oral mucosa for the detection of oral lesions. In addition, biological material was collected from labial and buccal mucosa for microbiological analysis. Oral mucositis was observed in only 5 (29.4%) patients. Microbiological analysis revealed a reduced number of potentially pathogenic microorganisms, such as coagulase-negative staphylococci (47%), Candida albicans (35.3%), Klebsiella pneumoniae (5.9%), enteropathogenic Escherichia coli (5.9%), and Stenotrophomonas maltophilia (5.9%). Patients with oral mucositis showed a higher frequency of coagulase-negative staphylococci (80%) when compared with patients with normal oral mucosa (33.3%). In conclusion, the results of the present study suggest that the prophylactic use of 0.12% chlorhexidine gluconate reduces the frequency of oral mucositis and oral pathogens in children with ALL. In addition, the present findings suggest a possible relationship between coagulase-negative staphylococci and the development of oral mucositis.