Guanidine‐based polymers have emerged as potential candidates for antibacterial agents and materials due to their high antibacterial property, no cytotoxicity, no bacterial resistance, and so on. A novel antimicrobial poly(methacryl guanidine hydrochloride)‐block‐polystyrene‐block‐poly(methacryl guanidine hydrochloride) (PMAGH‐b‐PS‐b‐PMAGH) is electrospun to ribbon‐like microfiber films for the first time. The electrospinning process parameters affecting structure and morphology of the ribbon‐like microfibers, including concentration, voltage, and feeding rate are systematically investigated. The mechanism inducing the formation of ribbon‐like fibers with a positively charged polymer is proposed and verified. PMAGH‐b‐PS‐b‐PMAGH microfiber films can inhibit 96% of Escherichia coli and 92% of Bacillus subtilis, and are not cytotoxic to mouse embryonic fibroblasts, but instead, provide a larger surface area for cell adherent growth.