Pleuromutilin is an antibiotic diterpenoid made by Clitopilus passeckerianus and related fungi, and it is the progenitor of a growing class of semi-synthetic antibiotics used in veterinary and human medicine. To harness the biotechnological potential of this natural product class, a full understanding of its biosynthetic pathway is essential. Previously, a linear pathway for pleuromutilin biosynthesis was established. Here we report two shunt pathways involving Pl-sdr and Pl-atf that were identified through the rational heterologous expression of combinations of pleuromutilin biosynthetic genes in Aspergillus oryzae. Three novel pleuromutilin congeners were isolated, and their antimicrobial activity was investigated, alongside that of an additional derivative produced through a semi-synthetic approach. It was observed that the absence of various functional groups – 3 ketone, 11 hydroxyl group or 21 ketone – from the pleuromutilin framework affected the antibacterial activity of pleuromutilin congeners. This study expands our knowledge on the biosynthesis of pleuromutilin and provides avenues for the development of novel pleuromutilin analogues by combining synthetic biology and synthetic chemistry.