Introduction: Antimicrobial resistance in bacterial pathogens is associated with high morbidity and mortality. We aimed to evaluate antibiotic resistance and β-lactamase production in clinical isolates of a tertiary care hospital in Central India. Materials and Methods: Clinical isolates (n=6472 isolates) from patients with infection were identified using standard microbiological techniques. Antibiotic susceptibility testing was performed according to the CLSI guidelines using the Kirby-Bauer disc diffusion technique. AmpC production in Enterobacteriaceae isolates was tested in screening test. Cloxacillin combined disc diffusion test was performed using cefoxitin disc with and without cloxacillin. Metallo-β-lactamase production in Enterobacteriaceae isolates was tested in screening test. Non-fermenting Gram-negative isolates were tested by combined disc test using imipenem and imipenem-EDTA discs. Results: Most bacteremia cases were caused by Staphylococcus aureus (43.13%), non-fermenting spp. (27.44%) and coagulase-negative staphylococci (11.76%). Escherichia coli (55.85%) was the main cause of urinary tract infection followed by Acinetobacter spp. (11.71%) and Klebsiella pneumoniae (10.36%). No resistance to linezolid was seen in Gram-positive isolates. Frequency of vancomycin-resistance was about 9% in Enterococcus spp. Methicillin resistance was seen in 19% of S. aureus isolates. Enterobacteriaceae and Citrobacter freundii isolates were completely resistant to aminopenicillin, first-and second-generation cephalosporins and cefamycin. Moreover, Klebsiella isolates were resistant to aminopenicillin. Enterobacteriaceae isolates showed resistance to aminopenicillin (89.87%), cephalosporins (54-90%) and cephamycin (37-45%). E. coli isolates were sensitive to piperacillintazobactam (87-96%) and imipenem (99.68-100%). Extended spectrum β-lactamase production was seen in 166 Enterobacteriaceae isolates (30.24%). AmpC production was seen in 15 (2.73%) Enterobacteriaceae isolates. Total β-lactamase production was found in 19.23% of the isolates. The frequency of β-lactamase production was highest in K. pneumoniae (51.67%). Conclusions: It is necessary to monitor drug resistance and β-lactamase production. Moreover, it is recommended to perform routine β-lactamase testing in microbiology laboratories for determining prevalence of antibiotic resistance and controlling their spread.