The textile industry’s reliance on synthetic dyes contributes significantly to pollution, highlighting the need for sustainable alternatives like biopigments. This study investigates the production and application of the biopigment prodigiosin, which was produced by Pseudomonas putida with a yield of 1.85 g/L. Prodigiosin was prepared under acidic, neutral, and alkaline conditions, resulting in varying protonation states that influenced its affinity for cotton and polyester fibers. Three surfactants (anionic, cationic, non-ionic) were tested, with non-ionic Tween 80 yielding a promising color strength (above 4) and fastness results with neutral prodigiosin at 1.3 g/L. Cotton and polyester demonstrated good washing (color difference up to 14 for cotton, 5 for polyester) and light fastness (up to 15 for cotton, 16 for polyester). Cellulose acetate, used in the conventional printing process as a thickener, produced superior color properties compared to commercial thickeners. Neutral prodigiosin achieved higher color strength, and cotton fabrics displayed halochromic properties, distinguishing them from polyester, which showed excellent fastness. Prodigiosin-printed samples also exhibited strong antimicrobial activity against Pseudomonas aeruginosa and retained halochromic properties over 10 pH cycles. These findings suggest prodigiosin as a sustainable dye alternative and pH sensor, with potential applications in biomedical materials, such as antimicrobial and pH-responsive wound dressings.