Parthenium hysterophorus L., also known as congress grass or carrot grass, is a widely distributed invasive weed which is a big challenge for sustainable agriculture. For the past several years, a number of attempts have been made to evaluate its medicinal properties. The present study aimed to evaluate the antimicrobial activity of the water-soluble ash extract of P. hysterophorus against different bacterial and fungal pathogens. The disc diffusion method was used to measure zone of inhibition (ZOI) whereas broth microdilution method was used to evaluate minimum inhibitory concentration (MIC) of the extract. The extract was found most effective against Staphylococcus aureus with a ZOI value of 33.5 mm at 33 mg/mL concentration when compared with ofloxacin (ZOI = 39.5 mm at 5 μg/disc). The extract was also found active against Salmonella enterica, Staphylococcus epidermidis and Escherichia coli with ZOI values of 25.5, 32.5 and 25.5 mm, respectively. The present study revealed that the ash extract of P. hysterophorus has potential to inhibit various bacterial species. However, the tested fungal pathogens like Candida albicans were found unaffected after treating with the ash extract.