Bacterial single-stranded (ss) DNA-binding proteins (SSBs) facilitate DNA replication, recombination, and repair processes in part by recruiting diverse genome maintenance enzymes to ssDNA. This function utilizes the C-terminus of SSB (SSB-Ct) as a common binding site for SSB's protein partners. The SSB-Ct is a highly conserved, amphipathic sequence comprising acidic and hydrophobic elements. A crystal structure of Escherichia coli exonuclease I (ExoI) bound to a peptide comprising the E. coli SSB-Ct sequence shows that the C-terminal-most SSB-Ct Phe anchors the peptide to a binding pocket on ExoI and implicates electrostatic binding roles for the acidic SSB-Ct residues. Here, we use SSB-Ct peptide variants in competition experiments to examine the roles of individual SSB-Ct residues in binding ExoI in solution. Altering the C-terminal-most Pro or Phe residues in the SSB-Ct strongly impairs SSB-Ct binding to ExoI, confirming a major role for the hydrophobic SSB-Ct residues in binding ExoI. Alteration of N-terminal SSB-Ct residues leads to changes that reflect cumulative electrostatic binding roles for the Asp residues in SSB-Ct. The SSB-Ct peptides also abrogate SSB stimulation of ExoI activity through a competitive inhibition mechanism, indicating that the peptides can disrupt ExoI/SSB/ssDNA ternary complexes. Differences in the potency of the SSB-Ct peptide variants in the binding and nuclease inhibition studies indicate that the acidic SSB-Ct residues play a more prominent role in the context of the ternary complex than in the minimal ExoI/SSB-Ct interaction. Together, these data identify roles for residues in the SSB-Ct that are important for SSB complex formation with its protein partners.