Chronic pelvic pain (CPP) is a complex condition with a high economic and social burden. Although it is usually treated with botulinum neurotoxin type A (BoNT/A) injected into the pelvic floor muscles (PFM), its effect on their electrophysiological condition is unknown. In this study, 24 CPP patients were treated with BoNT/A. Surface electromyographic signals (sEMG) were recorded at Weeks 0 (infiltration), 8, 12 and 24 from the infiltrated, non-infiltrated, upper and lower PFM. The sEMG of 24 healthy women was also recorded for comparison. Four parameters were computed: root mean square ( RMS), median frequency ( MDF), Dimitrov’s index ( DI) and sample entropy ( SampEn). An index of pelvic electrophysiological impairment ( IPEI) was also defined with respect to the healthy condition. Before treatment, the CPP and healthy parameters of almost all PFM sides were significantly different. Post-treatment, there was a significant reduction in power (< RMS), a shift towards higher frequencies (> MDF), lower fatigue index (< DI) and increased information complexity (> SampEn) in all sites in patients, mainly during PFM contractions, which brought their electrophysiological condition closer to that of healthy women (< IPEI). sEMG can be used to assess the PFM electrophysiological condition of CPP patients and the effects of therapies such as BoNT/A infiltration.