This paper presents the detection of nanoparticles and microorganisms using a recently developed side-orifice-based resistive pulse sensor (SO-RPS). By decreasing the channel height of the detection section of the SO-RPS, the detection sensitivity was increased and an average signal to noise ratio (S/N) of about 3 was achieved for 100 nm polystyrene particles. It was also found that spherical particles generate symmetrical signals. Algae with irregular shapes generate signals with more complex patterns. A scatter plot of signal magnitude versus signal width was proven to be reliable for differentiating bacteria from the nanoparticles and two types of algae. The side orifice for detecting heterogeneous nanoparticles and microorganisms is advantageous to avoid orifice clogging and the large flow resistance.