Bacterial endometritis refractory to appropriate antibiotic treatment may involve an biofilm. The production of an extrapolymeric substance, which are key components of the biofilm matrix, results in reduced host recognition and antibiotic tolerance. The goal of this study was to determine the in vitro effectiveness of dimethyl sulfoxide (DMSO), hypochlorous acid, ozone, or an antimicrobial peptide mimic (APM) at disrupting a preformed biofilm and evaluate the bacterial killing of Gram-negative isolates from the equine uterus. Dimethyl sulfoxide and the APM reliably eliminated preformed biofilm and reduced colony forming units (CFUs) of Escherichia coli and Klebsiella pneumoniae but were only effective against 10%–75% of the Pseudomonas aeruginosa isolates. The minimum biofilm eradication concentration (MBEC) for DMSO at disrupting an E. coli biofilm was 30% vol/vol, and the concentration for DMSO to reduce CFUs was 15% vol/vol for K. pneumonia. The MBEC concentration for AMP for reducing biofilm mass was 0.5% of the as directed solution for E. coli and K. pneumoniae. The MBEC concentration of AMP for reducing CFUs was 0.5% of the commercial solution for K. pneumoniae. Ozone and the two hypochlorous agents were not reliably able to reduce biofilm or kill any of the Gram-negative organisms tested. These bacterial species-specific results stress the importance of acquiring organism identification when developing a treatment plan for bacterial endometritis because not all treatments are effective against every bacterial species.