We present a novel and fully automated approach that combines an image-basedfuzzy logic controller (FLC) to precisely positionpre-selectedmicrofluidic picoliter droplets containing cultured bacteria with nonlinear microscopy for detailed analysisof droplet content. Using coherent anti-StokesRaman scattering (CARS) at the aliphatic CH-stretchingvibration of methylenegroups at 2841 cm−1 and two-photonexcited fluorescence (TPEF) microscopy, time series and z-stackimages of up to 50droplets were recorded. The autosampler adjusts the pressures at the inlet and outlet of the microfluidic chip in bright fieldmode to position a droplet in the center of the region of interest. Then, the pressures are kept constant, and CARS and TPEFimages are acquired in laser scanning mode for up to 60 s. Once the laser scanning image acquisition is complete, the nextdroplet is automatically processed. The software-basedintegration of the droplet autosampler and CARS/TPEF laser scanningmicroscopy is a powerful tool for fully automated investigation of microbial dynamics at the single-dropletlevel and providesinsights into bacterial morphology and motility, offering diffraction-limitedspatial resolution and a medium throughput ofthousands of droplets per day.