Despite numerous studies on nanofluids in microchannel heat sinks (MCHSs), they are not yet commercialized due to long-term stability issues and high maintenance costs. Therefore, this study explores the impact of nanofluids and nanoparticle clustering on single-phase convective heat transfer inside microchannels under laminar conditions. Water and commercially available water-based nanosuspensions, including Al2O3-water (30–60 nm), TiO2-water (5–30 nm), and polystyrene-water (50 nm), are circulated through silicon MCHS having rectangular channels integrated into a closed flow loop. To assess the in situ and real-time nanoparticle clustering during heat transfer experiments, Light Extinction Spectroscopy (LES) is applied as a non-intrusive measurement technique on nanofluids without any fluid sampling. Our findings reveal the appearance of nanofluid discoloration with no measurable increase in heat transfer coefficient. This unexpected change is attributed to the interplay of abrasion, erosion, and corrosion phenomena, likely triggered by the clustering of nanoparticles within the silicon microchannels—a novel insight into the complex dynamics of nanofluid behavior (an increase in the De Brouckere mean diameter from 11 nm to 107.3 nm over a 2.5 h period for TiO2 nanoparticles). The resulting material loss could not be mitigated by altering the nanoparticle material, which may impede heat transfer enhancement under tested conditions.