Poly(dimethylsiloxane) (PDMS) has become the material of choice for fabricating microfluidic channels for lab-on-a-chip applications. Key challenges that limit the use of PDMS in microfluidic applications are its hydrophobic nature, and the difficulty in obtaining stable surface modifications. Although a number of approaches exist to render PDMS hydrophilic, they suffer from reversion to hydrophobicity and, frequently, surface cracking or roughening. In this study, we describe a one-step in-mould method for the chemical modification of PDMS surfaces, and its use to assess the ability of different surfactants to render PDMS surfaces hydrophilic. Thin films of ionic and non-ionic surfactants were patterned into an array format, transferred onto silicone pre-polymer, and subsequently immobilized onto the PDMS surface during vulcanization. The hydrophilicity of the resulting surfaces was assessed by contact angle measurements. The wettability was observed to be dependent on the chemical structure of the surfactants, their concentration and interactions with PDMS. The morphology of modified PDMS surfaces and their change after wetting and drying cycles were visualized using atomic force microscopy. Our results show that while all surfactants tested can render PDMS surfaces hydrophilic through the in-mould modification, only those modified with PEG-PDMS-PEG copolymer surfactants were stable over wetting/dying cycles and heat treatments. Finally, the in-mould functionalization approach was used to fabricate self-driven microfluidic devices that exhibited steady flow rates, which could be tuned by the device geometry. It is anticipated that the in-mould method can be applied to a range of surface modifications for applications in analytical separations, biosensing, cell isolation and small molecule discovery.