Regenerated silk fibroin (RSF) is an emerging material derived from natural silk. Thin RSF films are transparent, biocompatible and biodegradable, which makes them suitable for many applications, such as flexible/conformal and transient electronics, bioresorbable devices, bioresists for lithography, and edible food protective coatings. To realize these applications, controlling and tuning the properties of RSF films is required to fully exploit their unique mechanical, optical, and degradation properties. Here, a new approach for tuning these properties is presented based on inducing rapid molecular structure transformations in fibroins via microwave heating. Transparent RSF films were post-treated by microwave irradiation, resulting in the transition of amorphous silk fibroin structure to a more α-helix dominant secondary structure. By increasing the microwave irradiation duration, an increase of helix secondary structure was observed. We use amide-I band Fourier-transform infrared spectroscopy (FTIR) of the films to characterize the secondary structure of fibroins. Moreover, we show that silicon substrates coated with 100 nm thick RSF films by spin casting, exhibit higher stability in water after microwave irradiation for up to 10 minutes, confirming a conformational change in the RSF secondary structure towards more stable α-helical rich motifs. Our results show that microwave treatment can be a new high throughput approach for tailoring the properties and structure of functional RSF-based films in a scalable and sustainable manufacturing process, when compared to other post processing techniques.