To address the need for new antibacterials, a number of bacterial genomes have been systematically disrupted to identify essential genes. Such programs have focused on the disruption of single genes and may have missed functions encoded by gene pairs or multiple genes. In this work, we hypothesized that we could predict the identity of pairs of proteins within one organism that have the same function. We identified 135 putative protein pairs in Bacillus subtilis and attempted to disrupt the genes forming these, singly and then in pairs. The single gene disruptions revealed new genes that could not be disrupted individually and other genes required for growth in minimal medium or for sporulation. The pairwise disruptions revealed seven pairs of proteins that are likely to have the same function, as the presence of one protein can compensate for the absence of the other. Six of these pairs are essential for bacterial viability and in four cases show a pattern of species conservation appropriate for potential antibacterial development. This work highlights the importance of combinatorial studies in understanding gene duplication and identifying functional redundancy.