Bacterial infections caused by multidrug resistant organisms are a major global threat. There is still a knowledge gap on this situation in the Northern Region of Ghana. This study determined the prevalence and resistance profile of bacterial infections. It also identified factors associated with multidrug resistance in the study area. This was a retrospective cross-sectional design and it analyzed data from the samples received at the Tamale Zonal Public Health Reference Laboratory from June 2018 to May 2022. The data were analyzed using the R software version 4.2.0. Univariate and multivariable binary logistic regression analyses were used to determine the factors associated with multidrug resistance. The samples included all specimen types possible. The specimens were collected for the purpose of clinical bacteriology diagnostics. Overall a total of 1222 isolates were obtained. The three (3) main bacteria responsible for infections were: Klebsiella spp. (27%), Moraxella spp. (22%), Escherichia spp. (16%). High resistance levels were found against the tested antibiotics and about 41.60% of the bacterial strains isolated were multidrug resistant. Hospitalization was associated with multidrug resistance in univariate (COR 1.96; 95% CI 1.43–2.71; P-value < 0.001) and multivariable analyses (AOR 1.78; 95% CI 1.28–2.49; P-value < 0.001). There is the need for further research on the molecular epidemiology of antibiotic resistance genes in the study area to effectively control the spread of multidrug resistant pathogens. In addition, efforts to build the capacity of health professionals on infection prevention and control as well as diagnostic and antimicrobial stewardship needs urgent attention.