Introduction: Antimicrobial resistance is a rising global public health threat. Knowledge on the circulating pathogens in a particular area and their antibiotic resistance profile is essential to direct clinicians on the rational antibiotic prescribing. The study was conducted to determine the microbial isolates and antibiotic susceptibility profiles of pathogens from a range of clinical samples in a tertiary hospital in Edo Central senatorial district in Edo state, Nigeria. Methods: The study was a retrospective analysis of microbiological isolates from clinical specimens collected between January 2016 and December 2019, using standard techniques from out-patient clinic attendees. Chi-square test was used to compare the association of type of bacterial isolates with patients’ sex, with the level of significance p set as <0.05. Prevalence rates of bacterial isolates and Resistance rates were calculated for each antibiotic used in microbiological culture. Results: Out of 3,247 clinical specimens processed, 994 (30.6%) showed microbial growth with 436 (43.9%) as gram-positive and 558 (56.1%) gram-negative bacterial isolates. Escherichia coli made up 286 (28.8%) of all isolates. Resistance to common antibiotics including cotrimoxazole, Tetracycline, Erythromycin and Cloxacillin were high for both microbial groups. Sensitivity to carbapenems, nitrofurantoin, and cephalosporins was high for gram-negative bacteria. Gram-positive bacteria exhibited high sensitivity to carbapenems and cephalosporins. Conclusion: High rates of resistance to common antibiotics were observed for gram-positive and gram-negative isolates. Hospital pharmacies and treatment guidelines should be made to reflect the current patterns of resistance to available antibiotics.