I present a technique to study the (dis)charging of organic semiconductor films at microscopic scales, and in various environments, using an optical tweezers-based method combined with fluorescence spectroscopy. The 1 μm silica spheres were coated with either pristine organic semiconductor or a donor-acceptor blend, trapped using optical tweezers, and their fluorescence was measured concurrently with the effective surface charge. The effective surface charge in uncoated silica spheres suspended in water was a factor of ∼70 higher as compared to that from similar spheres in a nonpolar toluene. In contrast, the coated silica spheres exhibited low effective charge densities in both environments, which is indicative of minimal interaction of organic semiconductors under study with these environments. This serves as a proof-of-principle experiment towards systematic studies of nanoscale photoinduced charge-based interactions between organic semiconductor molecules, with a resolution down to an elementary charge, and depending on the dielectric environment.