DNA and RNA strand exchange is a process of fundamental importance in biology. Herein, we used a FRET-based assay to investigate, for the first time, the stand exchange kinetics of natural DNA, natural RNA, and locked nucleic acid (LNA)-modified DNA sequences in vitro in PBS in the absence or presence of molecular additives and macromolecular crowders such as diethylene glycol dimethyl ether (deg), polyethylene glycol (peg), and polyvinylpyrrolidone (pvp). The results show that the kinetics of strand exchange mediated by DNA, RNA, and LNA-DNA oligonucleotide sequences are different. Different molecular crowders further affect the strand displacement kinetics, highlighting the complexity of the process of nucleic acid strand exchange as it occurs in vivo. In a peg-containing buffer, the rate constant of displacement was slightly increased for the DNA displacement strand, while it was slightly decreased for the RNA and the LNA-DNA strands compared with displacement in pure PBS. When we used a deg-containing buffer, the rate constants of displacement for all three sequences were drastically increased compared with displacement in PBS. Overall, we show that interactions of the additives with the duplex strands have a significant effect on the strand displacement kinetics and this effect can exceed the one exerted by the chemical nature of the displacement strand itself.