The goal of this study is to explore a new strategy to improve the mechanical and hydrophobic properties of bacterial cellulose (BC) mats. The present work is the first to report the preparation of in situ self-assembled BC nanocomposites using electrospun hydrophobic poly(lactic acid) (PLA) or PLA/lipids (PLA/Lip) nanofiber mats as foundation for BC nanofiber growth. Adding electrospun PLA mats to the BC culture media led to a two-fold increase in toughness with a 52% increase in elongation of the nanocomposites with regard to BC. The incorporation of electrospun PLA and PLA/Lip nanofiber mats lowered the moisture regain and water vapor transmission of BC nanocomposites relative to pure BC mats. The interfacial bonding between the individual components of a nanocomposite is a key factor for the improvement of composite strength, stiffness, and barrier properties; thus additional strategies to improve interaction between hydrophilic BC and hydrophobic PLA fibers need to be explored.