Aims: Over the decades, antibiotic resistance has become a cross-border public health problem. This calls for the profiling of microorganisms, particularly bacteria implicated in antibiotic resistance, in order to improve clinical practice and reduce the incidence of therapeutic failure in the treatment of infectious diseases. Study Design: We conducted a retrospective cross-sectional study. Place and Duration of the Study: The study was made at the bacteriology laboratory at the Yaounde University Teaching Hospital, Cameroon during the period between January 2016 and June 2021. Methodology: All bacterial strains from the following biological fluids were included: blood, stool, urine, suppurations, probe tip and catheter tip. The antibiotic susceptibility of isolates was collected from the registers of the said laboratory. The data were encoded in Censuses and Survey Procession Software (CSPro) version 7.3 and analysed using the Statistical Package for Social Science (SPSS) version 25. Graphs and figures were made using Excel 2016 spreadsheet software. Results: A total of 1071 bacteria were enrolled in 955 patients. The age group most represented was 0-5 years (34.6%). Most of the isolates came from a blood sample. Among the isolates, Coagulase-negative Staphylococci (18.5%), Escherichia coli (17.7%), Staphylococcus aureus (14%) and Klebsiella pneumoniae (11.2%) were the most common. A total of 1071 bacteria were enrolled in 955 patients. The age group most represented was 0-5 years (34.6%). Most of the isolates came from a blood sample. Among the isolates, Coagulase-negative Staphylococci (18.5%), Escherichia coli (17.7%), Staphylococcus aureus (14%) and Klebsiella pneumoniae (11.2%) were the most represented. Between 2016 and 2020, almost remarkable resistance was observed to the class of penicillins (78% to 83%), cephalosporins (44% to 61%) and quinolones (43% to 100%) for Escherichia coli. For Staphylococcus aureus, resistance changes range from 68% to 77% for the penicillin class. Klebsiella pneumoniae showed an evolution ranging from 11% to 19% for aminosides. Conclusion: Although not all isolates showed a change in the level of resistance to all antibiotics that are frequently used in our study population, Nevertheless, it is important for national public health actors to establish active surveillance of antibiotic and even antimicrobial resistance and to implement a guide to the proper use of antibiotics for health professionals, and the community.