Filamentous cyanobacteria develop heterocysts in response to deprivation for combined nitrogen under aerobic conditions. The most prominent structural change in heterocysts is the biosynthesis of an envelope that restricts gas permeability, providing an appropriate micro-oxic environment for N2 fixation inside. The additional thickness of the differentiated cells, when compared to vegetative cells, makes filamentous cyanobacteria an attractive biological system to investigate cellular response against femtosecond laser processing. By irradiating the cyanobacterial filaments with 120 fs, 795 nm, 1 kHz pulses focused through a 100× microscope objective with a numerical aperture of 0.85, we have determined that the pulse energy threshold for an apparent disruption of the cell wall of vegetative cells is 13 ± 4 nJ per pulse. A further increase in the pulse energy to 43 ± 13 nJ causes the complete removal of vegetative cells. In contrast, the pulse energy threshold has to be augmented about three-fold for heterocyst envelope disruption or two-fold for complete removal of heterocysts. We propose that the singular cross-linked structure of the glycolipid multilayer of the envelope, required to restrict gas permeability, accounts for the remarked difference in the ablation energy threshold between vegetative cells and heterocysts.