Xylella fastidiosa is a plant pathogen that threatens a US$ 4.6 billion worldwide wine and citrus industry. Monitoring its presence and distribution in plants and vectors is crucial for designing control strategies, as well as for understanding its ecological role and fate. We developed two fluorescent oligonucleotide probes complementary to different regions of the 16S rRNA gene of X. fastidiosa. The specificity of the newly designed probes S-S-X.fas-0067-a-A-18 and S-S-X.fas-1439-a-A-18 was demonstrated using fluorescence in situ hybridization (FISH) for 12 Xylella isolates, 15 closely related microorganisms and three plant endophytes. These probes were used to detect and quantify X. fastidiosa in plant sap (average value of 2.9 ± 0.3 × 106 cells ml−1) from three different citrus orchards. In a second experiment, cells were quantified in honeydew (2.2 ± 0.2 × 104 cells ml−1) collected from the insect vector Bucephalogonia xanthophis during the acquisition access period on an infected plant. The number of pathogen cells retained or digested by the insect is 10 000 times greater than the estimated minimum value to ensure an efficient transmission. Polymerase chain reaction (PCR) amplification using specific primers with plant sap and honeydew samples, followed by sequencing, confirmed the presence of the plant pathogen. This is the first demonstration of FISH being used for environmental samples, such as plant sap and insect honeydew, to estimate the abundance of a plant pathogen during infection.