Single-stranded DNA binding (SSB) proteins are essential proteins of DNA metabolism. We characterized the binding of the bacteriophage T4 SSB, Escherichia coli SSB, human replication protein A (hRPA), and human hSSB1 proteins onto model miniforks and double-stranded-single-stranded (ds-ss) junctions exposing 3' or 5' ssDNA overhangs. T4 SSB proteins, E. coli SSB proteins, and hRPA have a different binding preference for the ss tail exposed on model miniforks and ds-ss junctions. The T4 SSB protein preferentially binds substrates with 5' ss tails, whereas the E. coli SSB protein and hRPA show a preference for substrates with 3' ss overhangs. When interacting with ds-ss junctions or miniforks, the T4 SSB protein, E. coli SSB protein, and hRPA can destabilize not only the ds part of a ds-ss junction but also the daughter ds arm of a minifork. The T4 SSB protein displays these unwinding activities in a polar manner. Taken together, our results position the SSB protein as a potential key player in the reversal of a stalled replication fork and in gap repair-mediated repetitive sequence expansion.