In the quest for barrier membranes for the prevention of post-surgical tissue adhesions, polymer matrices may provide a platform of biomaterials with versatile properties. However, the relationship between the anti-adhesion effects of different polymer matrices and their physicochemical and structural properties is not yet adequately understood. In a preclinical study using a rat cecum model, we directly compared the anti-adhesion potential of polyhydroxybutyrate (PHB) electrospun nanofibrous versus cast film matrices loaded with methylene blue (MB) as antioxidant adhesion inhibitor. PHB retained MB presumably forming MB-bioactivated matrices. In the preclinical study, quantitative morphologic assessment in addition to histopathologic and SEM examinations 14 days post-surgery indicated that plain PHB NFs and MB-PHB NFs, moderately enhanced cecal wall healing and inhibited adhesion formation. In contrast, reshaping PHB as cast films, significantly enhanced healing, reduced adhesion bands and prevented inter-visceral adhesions. Cast films also inhibited tissue attachment to the matrix recovered 14 days post-surgery. Both PHB matrix types reduced tissue inflammation. Despite tissue anti-adhesion potential of individual matrix components, modulation of the micro-architectural properties generated polymer barriers with varying tissue anti-adhesion and healing potentials, the MB-loaded cast film achieving the best outcome.