Polydiacetylenes (PDAs) are conjugative polymers that demonstrate color changes as a response to an external stimulus. In this study, 10,12-pentacosadiynoic acid (PCDA) was mixed with a supporting polymer including poly(ethylene oxide) (PEO) and polyurethane (PU), and the mixture solution was electrospun to construct fiber composites. The electrospun fibers were then photopolymerized using UV irradiation to produce PEO–PDA and PU–PDA nanofiber mats with a fiber diameter ranging from 130 nm to 2.5 μm. The morphologies of both PEO–PDA and PU–PDA nanofibers were dependent on electrospinning parameters such as the ratio of PCDA to PEO or PCDA to PU and the total polymer concentrations. Scanning electron microscopy images showed beaded fibers of PEO–PDA and PU–PDA at 2 and 18 w/v % concentrations, respectively. Smooth fibers were found when the solvent concentration was increased to 3.75 w/v % in PEO–PDA and 25 w/v % in PU–PDA fibers. Both PEO–PDA and PU–PDA nanofiber composites demonstrated excellent colorimetric responses to the presence of Escherichia coli ATCC25922 bacterial cells and the changes in pH as external stimuli. The nanofibers underwent a rapid colorimetric response when exposed directly to E. coli ATCC25922 grown on Luria–Bertani agar. The comparison between the PEO–PDA and PU–PDA suggested that the combination of PEO and PDA is favorable because it provides a sensitive response to the presence of E. coli. The results were compared with samples of a PDA polymer in the absence of a matrix polymer. The colorimetric response was similar when the PDA polymer and the PDA nanofiber composites were exposed to pH changes, and the color change was found to occur at pH 10 and enhanced at pH 11–13. The PDA-containing nanofiber composites showed stronger colorimetric responses than those of the PDA polymer only, suggesting their potential as biosensors and chemosensors.