Previous studies have reported that different phenothiazine derivatives have shown a broad spectrum of antibacterial, anticancer and antiplasmid activities. In the present study, we investigated the interactions of phenothiazine (phtz) and its N-methyl-substituted derivative, N-methylphenothiazine (N-Mephtz) with bovine serum albumin (BSA) and calf thymus DNA (ct-DNA) by fluorescence emission spectroscopy to examine their binding affinity towards these biomolecules. Considering that serum albumin is divided into three domains (I–III), with each domain containing two subdomains (A and B), we have also performed fluorescence competition experiments with site markers for BSA to locate the binding site of the investigated compounds to this biomolecule. Eosin Y was used as a marker for site I (subdomain IIA), while ibuprofen was a marker for site II (subdomain IIIA). The obtained results and the values of binding constants (K_A) have indicated that both phtz and N-Mephtz can interact with BSA and ct-DNA, whereby N-Mephtz has higher binding affinity towards these biomolecules. On the other hand, K_A values of both investigated compounds are lower in the presence of eosin Y, while only a slight change was observed in the presence of ibuprofen. These results indicated that the binding of the investigated compounds should be mainly located within site I of BSA, and that the tested compounds had to compete with eosin Y to bind to this protein.