To combat the increasing prevalence of antimicrobial resistance in bacterial pathogens, discovery of novel antimicrobial molecules is vital. In microbial communities, bacteria have developed various methods to increase their competitive advantage, including the production of antimicrobial molecules. We have exploited some of these antimicrobials to develop our current clinical antibiotics, however there are still many bacterial communities that have not yet been investigated for novel antimicrobials, including the human gastrointestinal microbiome. Due to the abundance and diversity of bacterial species in the gastrointestinal tract, competition in this environment is high, and microbiota species may benefit from the advantages provided by antimicrobial production. Genomic analysis shows that many of these species carry biosynthetic gene clusters that may encode for novel antimicrobial molecules, which can now be investigated in the laboratory due to recent advancements in culturing techniques that allow us to cultivate the previously “unculturable” members of the human gastrointestinal microbiota. This technique offers a promising approach in exploring a new source of therapeutics for the treatment and prevention of drug-resistant infections. In this study, we used a high throughput culturing method, to screen a panel of 287 commensal bacterial isolates for antimicrobial activity against eight multi-drug resistant strains of four species of gastrointestinal pathogens: Clostridiodes difficile, Escherichia coli, Enterococcus faecium, and Klebsiella pneumoniae. Of the isolates screened, 148 (52%) exhibited inhibition of at least one pathogen strain. From these isolates, 20 candidates that displayed inhibition of all four pathogen species were selected for further validation of inhibitory activity, which confirmed inhibition in six candidates. To determine mechanism of inhibition, the cell free supernatant of these candidates was used in a well-diffusion assay, however these exhibited variable results. Further investigation into the requirements for antimicrobial production in these commensal strains is required and will aid in the characterisation of the molecules responsible for inhibition, which could lead to development of novel antibiotics.