Globally, the burden of chronic wound infections is likely to increase due to the rising levels of bacterial resistance to antibiotics. In the United States of America alone, more than 6.5 million chronic wounds with evidence of bacterial infection are diagnosed every year. In addition, the polymicrobial environment in chronic wound infections has been observed from several studies as a risk factor for development of resistance to many antibiotics including the third generation cephalosporins currently used in Mbarara Regional Referral Hospital for treatment of chronic wound infections. Therefore the main objective of this study was to determine the prevalence of chronic wound isolates and their minimum inhibitory concentrations (MIC) against third generation cephalosporins. This study was a cross-sectional descriptive and analytical survey of bacterial isolates from chronic wound infection among 75 study participants admitted in the surgical ward of Mbarara Regional Referral Hospital (MRRH), a tertiary Hospital in Western Uganda. Standard laboratory bacterial culture and identification techniques as well as broth microdilution method were used to isolate, identify pathogens and test for MIC respectively. We found that 69/75 study participants had samples with bacterial growth and the most prevalent pathogens isolated were staphylococcus aureus (40.6%) and Klebsiella spp. (29%). Generally, most isolates were susceptible to cefoperazone + sulbactum 2 g (Sulcef) and ceftriaxone 1 g (Epicephin). The overall prevalence of isolates in chronic wound infection among patients admitted in the surgical ward of MRRH was 92% and the most prevalent isolates were Staphylococcus aureus, Klebsiella species and proteus species respectively. The observed MIC values were higher than the CLSI clinical breakpoint, implying a decreasing trend in susceptibility of chronic wound isolates to third generation cephalosporins.