Isolates obtained from laboratory surfaces were identified and characterized. Ten consecutive isolates were obtained from 30 sample surfaces of a University Science Laboratory in Edo State Nigeria in May, 2021. Swabs of surfaces from the laboratory were obtained aseptically. The sample swabs were streaked on MacConkey, eosin methylene blue, mannitol salt, and nutrient agar plates, respectively, and incubated appropriately. Distinct colonies were randomly obtained from culture plates and characterized phenotypically. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was used to analyze four isolates (40%) obtained by selection criteria. Susceptibility testing using antibiotics was performed for the identified isolates by Kirby-Bauer method for 15 antibiotics. Isolate characterization and identification of resistance determinants were determined using whole genome sequencing (WGS). Microorganisms identified included Leclercia adecarboxylata, Enterobacter hormaechei, Atlantibacter hermanii, and Stenotrophomonas maltophilia. Three identified isolates were antibiotics-resistant and were investigated by WGS. Resistance genes were found in all (100%) of the resistant laboratory isolates. The resistance determinants included β-lactamase genes, aminoglycoside modifying enzymes, qnr genes, sulfonamide, tetracycline, and trimethoprim resistance genes, respectively. Two isolates carried ESBL genes and blaCTX-M-15 was detected. Our study displays the dissemination of antibiotic resistance among isolates obtained from surface of a University Science Laboratory. To the best of our knowledge, we have reported the first genomic characterization of resistance to antibiotics in isolates obtained from surfaces of a University Science Laboratory in Nigeria.