Periodic monitoring of antibiotic susceptibility patterns in clinical settings is vital to ascertain the potency as well as re-establishing empirical therapy. This retrospective study aimed to evaluate the antibiotic susceptibility patterns of pathogens isolated from routine laboratory specimens at Ndola Teaching Hospital. A retrospective study was conducted on routine specimens received between May 2016 and July 2018. Specimens were cultured on standard media and Kirby-Bauer disc diffusion method was used for susceptibility testing in accordance with the Clinical and Laboratory Standard Institute's recommendations. A total of 693 specimens were analyzed, of which 65.9% (457) specimens came from inpatient departments and 49.1% (340) came from female patients. The commonest specimens were urine (58.6%), blood (12.7%) and wound swabs (8.5%), and the most common microorganisms were coliform (29.3%), Staphylococcus aureus (15.4%), coagulase negative Staphylococci (CoNS, 13.4%), and Escherichia coli (13%). The highest percentage of resistance to any particular antibiotic was co-trimoxazole (91.7%, 33) followed by nalidixic acid (75.2%, 279), norfloxacin (69.0%, 100), ceftazidime (55.7%, 185), nitrofurantoin (46.6%, 191), chloramphenicol (43%, 111) and ciprofloxacin (8.6%, 271). Furthermore, patient location had resistance effect on coliform (p = 0.014), CoNS (p = 0.031), Streptococcus species (p = 0.024) and Klebsiella species (p = 0.004) to nitrofurantoin, ceftazidime, nitrofurantoin and chloramphenicol, respectively. Besides coliform, resistance of Enterobacter species to ceftazidime and Proteus species to nalidixic acid were more from female patients. Generally, the most effective antibiotics were chloramphenicol and nitrofurantoin with addition of ceftazidime on blood pathogens and ciprofloxacin on wound swab pathogens. The common isolates were coliform, S. aureus, coagulase negative Staphylococci and Escherichia coli. The resistance of most bacteria to ceftazidime and nitrofurantoin were influenced by both gender and location. Our study presents a broad overview of the resistance profiles of bacterial isolates. However, more nosocomial prevalence and antibiogram studies on individual routine specimens are required to provide a more detailed picture of resistance patterns.