Background and objective: Asymptomatic bacteriuria (ASB) poses a serious health problem to pregnant women and fetuses. However, in most developing countries, routine screening for ASB and antimicrobial sensitivity test are rarely performed. This study, therefore, aimed to determine the best diagnostic method for routine screening of ASB and antimicrobial susceptibility pattern among pregnant women attending an antenatal clinic in the Ashanti Region of Ghana. Methods: Urine samples from 412 pregnant women between the ages of 16 and 45 years-old attending antenatal clinic at Anglogold Ashanti Health Foundation Hospital and Ellolab Diagnostic Centre were screened for ASB by microscopy, dipstick urinalysis and bacteria culture. Susceptibility of the positive isolates were assessed against commonly used antimicrobial agents, adopting the disc diffusion test method. Results: Of the 412 pregnant women screened, 72 tested positive for ASB by the urine culture method, which translates into an overall prevalence of 17.5%. There was no association between age, marital status, occupation, parity, educational background nor duration of pregnancy with ASB (p > 0.05). Additionally, dipstick urinalysis was found to be a better diagnostic method than microscopy. The most isolated bacteria were Escherichia coli (62.5%) and Klebsiella pneumoniae (30.6%), and nitrofurantoin and nalidixic acid were the most effective antimicrobial agents. Conclusions: Routine urine culture and antimicrobial susceptibility test should be carried out on all pregnant women attending antenatal clinic to detect possible ASB and prescribe appropriate drugs, such as nitrofurantoin and nalidixic acid, to prevent any related complications. However, in health centers that lack bacterial culture facilities, dipstick urinalysis should be the preferred screening option.