Nano-structured black silicon (bSi) was used as a substratum for the construction of a microfluidic device to test the bactericidal action of this nano-textured surface against Pseudomonas aeruginosa bacteria. A narrow 15 µm high and 1 cm wide flat flow channel was constructed that allowed the bacteria to come into contact with the bactericidal nano-spikes present on the surface of the bSi. The narrow channel within the device was designed such that a single layer of bacterial cells could reside at any given time above the bSi substratum during flow. The large 1 × 2 cm² surface area of the bSi was shown to be efficient in being able to kill the bacterial cells, achieving an approximate 99% killing efficiency. The flow rate required to fill the bSi chamber was found to be 0.1 µL s⁻¹, with a 10 min equilibration time being allowed for the bacterial cells to interact with the bSi surface. Complete rupturing of E. coli cells was achieved after 15 cycles, allowing the effective release of cellular proteins from within the bacterial cells (65.2 µg mL⁻¹ from 3 × 10⁸ cells per mL). The channel was then able to be re-used after washing of the cell with 10 successive cycles of sterile MilliQ water. Larger volumes of bacterial suspensions have the potential to be treated using a similar flow channel configuration if the dimensions of the flow channel are scaled accordingly. This bactericidal microfluidic device provides a novel platform for studies carried out under both static and dynamic (flow) conditions.