Bacteriophages represent a remarkably versatile probe for biosensing and a key component of a new class of bioactive surfaces. Chemical immobilization of bacteriophages is a key operation enabling such applications, yet despite this, rarely is a comparison made between immobilization chemistries or for multiple phages with the same parameters. Here, we report the immobilization of bacteriophages 44AHJD, P68, Remus, and gh-1 by physisorption and covalent cross-linking via a series of thiolated reagents: 11-mercaptoundecanoic acid (11-MUA), L-cysteine with 11-MUA, L-cysteine with glutaraldehyde, and dithiobis-(succinimidyl propionate). Surprisingly, phage purification protocols showed significant impact on the phage immobilization efficiency. Indeed, purification of phages by density gradient (CsCl) ultracentrifugation and centrifugal ultrafiltration was found to have a dramatic determinant effect on the quality of the immobilized layer. Surface densities of 160 ± 13.9 phage/μm2 were observed when careful phage purification was combined with 11-MUA self-assembled monolayer functionalization of the surface. High-resolution scanning electron microscopy enabled direct confirmation of immobilization, along with calculation of phage densities on the surface, and even resolution of phage capsid substructures.